Assays of the biochemical functions of G-protein beta-gamma subunits were used to assess the status of these proteins in ACTH-unresponsive mutant Y1 adrenal cortical cells. Analysis of the expression of the beta subunit chains with molecular probes identified a modest decrement of the beta2 gene product. However, in comparison with wild-type Y1 cells, the mutant clones displayed significantly diminished beta-gamma activity, indicating a mutation of the gene (s) or defect in post-translational modification. we have constructed baculo viral expression vectors and obtained significant levels of expression of G-protein beta-gamma dimers in Sf9 cells. Techniques were devised to purify the recombinant proteins to homogeneity, and their specific biochemical activities were assessed. The unmodified beta-gamma dimer interacts with identical affinity for alpha subunit, but diminished affinity for receptor. Thus, prenyl modification not only anchors the beta-gamma dimer to the plasma membrane, but also enhances the receptor interaction of the dimer. A novel mechanism for G-protein activation by in situ GTP synthesis catalyzed by nucleoside diphosphate kinase (NDK) was examined. While in vitro GTP formation using GDP-bound ARF protein was found, when we attempted to extend these observations to the signal transducing G-proteins, all effects of the NDK were found to be artifactual. These studies refute the proposed role for the nm-23 gene products as regulators of G-proteins.